Membrane and protein trafficking represent areas of high current interest and great excitment and promise in cell biology at present. The reason for this high current interest is that protein sorting and vesicular membrane traffic are involved in a wide array of important cellular processes, including protein secretion, biogenesis of membrane proteins and lysosomal enzymes, uptake of metabolites, peptide hormones and growth factors, regulation of cell surface receptors and transporters, and antigen presentation and recognition, to mention a few. Progress in understanding pathways of membrane traffic has been rapid in the last 5 years, but we still have a long way to go and many questions must be answered before we fully understand the mechanisms involved. Our interest for some time has centered on traffic along the secretory or exocytic pathway. During the previous grant period we have: 1) established the recycling itinerary of the 215kD receptor (Golgi to endosomes and back via coated vesicles); 2) determined that the distribution of these receptors within the Golgi complex differs among cell types with different routes of lysosomal enzyme traffic; 3) shown that agents which perturb lysosomal enzyme traffic cause a redistribution of receptors along their normal recycling pathway; 4) established that plasmalemmal transferrin receptors can visit cis and middle as well as trans Golgi cisternae during recycling; and 5) identified a 58kD protein that appears to represent a general marker for cis Golgi cisternae. During the next grant period we intend to pursue these problems further, and we have the following goals: 1) To extend our knowledge of the role of the 215kD Man6P receptor in lysosomal enzyme trafficking in terms of the mechanisms, pathways and compartments involved (e.g., during crinophagy and autophagy) and compare its role to that of the 46kD Man6P receptor; 2) To determine the routes and site of separation of regulated and constitutive exocytic traffic as well as the points of interface between exocytic and endocytic traffic in AtT-20 pituitary cells; 3) To characterize the newly-discovered 58kD cis Golgi protein in terms of the steps in its biosynthesis, the kinetics of its incorporation into the cis Golgi cisterna and its amino acid sequence; 4) To identify and characterize markers for other Golgi elements -- especially trans Golgi elements and the vesicles involved in ER to Golgi transport.